Hepatic Uptake of Bile Acids in Man

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1 Hepati Uptake of Bile Aids in Man FASTING AND POSTPRANDIAL CONCENTRATIONS OF INDIVIDUAL BILE ACIDS IN PORTAL VENOUS AND SYSTEMIC BLOOD SERUM Bo ANGELIN, INGEMAR BJORKHEM, KURT EINARSSON, and STAFFAN EWERTH, Departments of Mediine, Clinial Chemistry, and Surgery, Karolinska Institutet at Huddinge University Hospital, Stokholm, Sweden A B S T R A C T This investigation was undertaken in order to (a) haraterize the postprandial inflow of individual bile aids to the liver and (b) determine if peripheral venous bile aid levels always adequately reflet the portal venous onentration, or if saturation of hepati bile aid uptake an our under physiologial onditions. In five patients with unompliated holesterol gallstone disease, the umbilial ord was annulated during holeystetomy, and a atheter was left in the left portal branh for 5 to 7 d. The serum onentrations of holi aid, henodeoxyholi aid, and deoxyholi aid in portal venous and systemi irulation were then determined at intervals of 15 to 3 min before and after a standardized meal. A highly aurate and speifi gas hromatographi/ mass spetrometri tehnique was used. The sum of the fasting onentrations of the three bile aids averaged 14.4±4.13 Amol/liter in portal venous serum, and 2.44±.31 Amol/liter in peripheral venous serum. The estimated hepati frational uptake of holi aid was -9%, and those of henodeoxyholi aid and deoxyholi aid were 7-8%. This resulted in an enrihment of systemi bile aids in the dihydroxy bile aid speies. In response to a standardized meal, portal venous bile aid onentrations inreased two- to sixfold, with a peak seen 15-6 min after the meal. The maximum postprandial portal venous bile aid onentration averaged 43.4±6.12,umol/liter, and the orresponding onentration in peripheral serum was 5.22±.74 A.mol/liter. The estimated frational uptakes of the individual bile aids were not affeted by the inreased inflow to the liver. The peripheral venous onentrations of individual as Reeived for publiation 12 Marh 1982 and in revised form 9 June Dr. Angelin is the reipient of a researh fellowship from the Ernst Klenk Foundation. well as total bile aids were well orrelated with those in portal venous serum. The results (a) give a quantitation of postprandial bile aid inflow to the liver and (b) indiate that the hepati uptake system for bile aids in healthy man annot be saturated during maximal inflow of endogenous bile aids. Measurement of peripheral serum bile aids an thus give important information on the status of the enterohepati irulation. INTRODUCTION In man, the two primary bile aids, holi aid (C)' and henodeoxyholi aid (CD), together with the seondary bile aid, deoxyholi aid (D), undergo an effiient enterohepati irulation (1). Sereted as glyine or taurine onjugates by the liver, the bile aids are reabsorbed in the small intestine and returned to the liver in the portal vein. The portal venous inflow of bile aids is of regulatory importane for the hepati bile seretion, and for the onversion of holesterol to bile aids (1-4). Due to the effiient hepati uptake, fasting serum bile aid onentrations are generally low in peripheral irulation, in spite of a signifiant inflow of bile aids to the liver in the portal vein (5). In response to a meal, peripheral venous levels of serum bile aids inrease severalfold (6, 7). This is presumed to be the onsequene of an inreased absorption of bile aids, and thus an elevated portal venous load to the liver. By diret measurement of the individual bile aids in portal venous and systemi irulation we have shown that there are differenes in intestinal uptake and hepati learane of individual bile aids in fasting man (5). The relation between individual bile aid onentrations in portal venous and systemi irula- ' Abbreviations used in this paper: C, holi aid; CD, henodeoxyholi aid; D, deoxyholi aid. 724 J. Clin. Invest. - The Amerian Soiety for Clinial Investigation, In /82/1/724/8 $1. Volume 7 Otober

2 tion during a meal has not been studied previously in man. Thus it is not known if peripheral venous bile aid levels always adequately reflet the portal venous onentration, or if saturation of hepati bile aid uptake an our under physiologial onditions. To address these questions, we have determined the onentrations of C, CD, and D, using an aurate gas hromatographi/mass spetrometri tehnique, in portal venous and systemi blood serum of holeystetomized subjets fed a standardized meal. METHODS Patients. Five patients, one male and four females, were inluded in the study. They were all referred to the hospital beause of unompliated gallstone disease. The stones onsisted of >7% holesterol in all ases. None of the patients had a history of ommon dut stones, and they all had a normal operative holangiogram. Histologial examination of operative liver biopsies was normal in all ases. Basal data on the patients are presented in Table I. None of them had linial or laboratory evidene of diabetes mellitus, hyperlipidemia, or diseases affeting kidney or thyroid funtion. Exept for a slightly elevated serum bilirubin onentration in one of the patients, they all displayed normal liver funtion tests. None of the patients were on treatment with drugs. Experimental proedure. All patients had an uneventful holeystetomy under standardized onditions as desribed (5, 8). During laparotomy, the umbilial vein was identified and annulated as desribed (9). The atheter was inserted into the left portal branh, lose to the main stem, and filled with a heparin solution. There was no evidene of ompliations in any of the patients, and they all had a normal postoperative reovery and mobilization. A detailed desription of the surgial proedure, together with a disussion of the prevention of possible ompliations, has been published reently (9). After 5-7 d, when the patients had been given oral meals for 3-4 d, and their bowel habits were normal, they were studied in the morning after a 12-h fast. At 7:3 a.m., an indwelling needle was inserted into an anteubital vein, and two to three blood samples were drawn from peripheral and portal venous irulation in the fasting state. At 8: a.m., the patients were given a standardized breakfast, onsisting of 2 dl milk, two heese sandwihes, and 2 dl offee. The energy ontent of this meal is equivalent to 1,75 kj with arbohydrate, fat, and protein aounting for 33, 44, and 23% of total, respetively. 5 ml of blood were then drawn from the peripheral venous and portal venous atheters at intervals of 15 min for min. The portal venous atheter ould then be withdrawn without ompliations, and the patients were disharged from the hospital on the following day. All patients had given their informed onsent before surgery, and the ethial aspets of the study had been approved by the Ethial Committee of Karolinska Institutet. Analytial proedures. After lotting of blood at room temperature, serum was obtained by entrifugation and frozen at -2 C for subsequent analysis. To.5 ml of peripheral and.1 ml of portal venous serum, 2.5 ug of [2,2,3,4,4,-2H5] C, 2.5 jig of [2,2,3,4,4-2H5] CD, and 5. Ag of [11,11,12-2H3] D, dissolved in aetone, were added. The serum, with the added internal standards, was hydrolyzed with 1 M KOH at 11 C for 12 h. The alkaline solution was extrated three times with diethyl ether to remove most of the neutral steroids. The bile aids were then extrated from the aidified water phase with ethyl ether, methylated with diazomethane, and onverted into trimethylsilyl ether derivatives. The derivatives were analyzed by gas hromatography-mass spetrometry using an LKB 9 instrument equipped with a multiple ion detetor (MID) unit. A 1.5% SE-3 olumn was used, and the operating temperature was 23-27C. Our desribed mass fragmentographi tehnique (1) for determination of the individual serum bile aids, C, CD, and D, has been modified, using more speifi ions for eah bile aid derivative. As a onsequene, however, eah bile aid must be determined separately with our instrument (LKB 9). Thus, C was analyzed with two of the hannels foused on m/e 623 and m/e 628, orresponding to the M-15 peak in the mass spetrum of trimethylsilyl derivative of unlabeled and deuterium-labeled C, respetively. CD was analyzed with two of the hannels foused on mle 37 and 373, orresponding to the M-2 X 9 peak in the mass spetrum of trimethylsilyl derivative of unlabeled and deuterium-labeled CD, respetively. D was analyzed with two of the hannels foused on m/e 255 and 258, orresponding to the base peak in the mass spetrum of trimethylsilyl deriv- TABLE I Basal Data on the Patients Patient no. Sex' Age RBWI Bilirubini ASAT ALAT API grmo1/liter Mkat/liter 1 M F F F F Mean±SEM 62±3 14±5 11.1±2.5.4±.3.41±.2 3.2±.4 M, male; F, female. Relative body weight, alulated as weight (kg)/[height (m) - 1] x 1%. Normal range: bilirubin, 3.4 to 2.5; ASAT (aspartate aminotransferase) <.7; ALAT (alanine aminotransferase) <.7; AP (alkaline phosphatase) < 5.4. Hepati Uptake of Bile Aids in Man 725

3 ative of unlabeled and deuterium-labeled D, respetively. The onentrations of the individual bile aids were thus alulated from the ratios C/[2,2,3,4,4-2H5]C (traing at m/ e 623/traing at m/e 628), CD/[2,2,3,4,4-2H5]CD (traing at m/e 37/traing at m/e 373), and D/[11,11,12-2H3]D (traing at m/e 255/traing at m/e 258). Standard urves were used for eah bile aid. The relative standard deviation of the method, as alulated from dupliate samples, was 2-3%. RESULTS The sum of the fasting onentrations of C, CD, and D averaged 14.4±4.13 gmol/liter (mean±sem) in portal venous serum, and 2.44±.31,umol/liter in peripheral venous serum. With the exeption of patient 4, who had a relative dominane of C in both portal venous and peripheral serum, the onentrations of the three bile aids were roughly similar in the portal vein (Table II). In all ases, the ratio between portal venous and peripheral venous onentration was higher for C than for CD or D, indiating a more effiient hepati uptake of the trihydroxy bile aid. Patient 1, who displayed a slightly elevated serum bilirubin onentration, also had the lowest ratios between portal venous and peripheral venous onentrations for all three bile aids. However, the fasting serum bile aid onentrations of this patient were not inreased (Table II). In response to the meal, there was a rapid rise in the portal venous as well as the peripheral venous onentration of all three bile aids. In all patients, there was a tendeny to an earlier rise of the bile aid onentration in the portal vein than in the peripheral irulation. Two representative examples are seen in Figs. 1 and 2 (patients 2 and 4). A first peak onentration ourred simultaneously for all three bile aids within 15 to 6 min after the meal (Table III). Bile aid onentrations then dereased somewhat over a 3-6-min period, whereafter a seond peak (generally lower) ourred 9 min after the initial one. - The sum of the onentrations of C, CD, and D at the maximum of the first peak averaged Mmol/liter in portal venous and 5.22±.74 ;Lmol/liter in peripheral venous serum, respetively (Table III). The ratio between portal venous and peripheral venous onentration was higher for C than for CD or D also during maximal inflow of bile aids. Again, patient 1 displayed lower ratios, whereas his peripheral venous bile aid levels were not learly distinguishable from those of the others. The data thus indiated an effiient uptake of all three bile aids both in the fasting and in the postprandial state. To make an estimation of the frational uptake of the individual bile aids, we have to assume that the onentration of the bile aids in hepati venous blood serum is similar to that in the peripheral irulation. This assumption was validated by studies in five healthy volunteers, undergoing hepati venous atheterization as part of an investigation on arbohydrate metabolism (f. 11). As seen in Table IV, simultaneously determined hepati venous and arterial onentrations of C, CD, and D were in all ases exept one almost idential. Furthermore, as we ould not diretly determine blood flow, we have to assume a onstant ratiq of 1:3 between the hepati arterial and the portal venous flow (f. 5). In the fasting state, this estimated uptake was -85% for C and lower, -7%, for CD and D (Table V). Patient 1 had a learly redued frational uptake of all three bile aids. The orresponding values were similar-or even slightly higher-for all three bile aids during maximal postprandial inflow. The estimated frational uptake of C was again higher than that of CD or D. Although there was some sattering of the data points, the peripheral venous onentration of an in- TABLE II Fasting Conentrations of Individual Bile Aids in Portal Venous and Peripheral Venous Serum of Five Choleystetomized Patients' Choli aid Chenodeoxyholi aid Deoxyholi aid Patient no. P V P/V P V P/V P V P/V ,umol/liter glmol/liter jamollliter Aimollliter pmol/liter prnol/liter Mean±SEM 6.13± ± ± ±.63.64±.6 4.7±.9 4.9± ±.11 4.±1. a P, portal venous onentration; V, peripheral venous onentration; P/V, ratio between portal venous and peripheral venous serum onentration B. Angelin, I. Bjdrkhem, K. Einarsson, and S. Ewerth

4 2~ A - B 15 a ;1._i a S Thw (mhi) FIGURE 1 Conentrations of (A) holi aid, (B) henodeoxyholi aid, and (C) deoxyholi aid in portal venous (-) and peripheral venous () serum in response to a standardized meal given at time (patient 2). Bile aid onentration (miromoles/liter). dividual bile aid refleted the simultaneous portal venous onentration (Fig. 3 A-C). As a onsequene of the lower uptake of the bile aids in patient no. 1, the relationship was somewhat different in this ase. The relative omposition of the individual bile aids varied both between the different subjets and as a 3.i 5IA 3[ 25[ 2F I15 B ~ ~ Th (mni) FIGURE 2 Conentrations of (A) holi aid, (B) henodeoxyholi aid, and (C) deoxyholi aid in portal venous () and peripheral venous (-) serum in response to a standardized meal given at time (patient 4). Bile aid onentration (miromoles/liter). Hepati Uptake of Bile Aids in Man 727

5 TABLE III Maximal Postprandial Conentrations of Individual Bile Aids in Portal Venous and Peripheral Venous Serum of Five Choleystetomized Subjets' Choli aid Chenodeoxyholi aid Deoxyholi aid Patient no. Time P V P/V P V P/V P V P/V min jmol/liter jumol/liter prmol/liter /umol/liter umol/liter pmol/liter Mean±SEM ±4.17 ±.38 ±4.1 ±.7 ±.26 ±1.7 ±3.71 ±.36 ±1.7 Time, time of maximal onentration (first peak) in relation to meal; P, portal venous onentration; V, peripheral venous onentration; P/V, ratio between portal venous and peripheral venous serum onentration. onsequene of the meal. Nevertheless, the sum of C, CD, and D ("total bile aids") in peripheral irulation orrelated with the total onentration in the portal vein (Fig. 4). DISCUSSION The tehnique used for determination of the serum onentrations of C, CD, and D used in the present work is very similar to the one desribed previously by us (5, 7, 1). The inlusion of one deuterium-labeled internal standard for eah of the three bile aids, as well as the monitoring of speifi ion pairs for eah bile aid, would theoretially further improve the auray of the method (1). It should be pointed out TABLE IV Fasting Conentrations of Individual Bile Aids in Hepati Venous and Peripheral Arterial Blood Serum of Five Healthy Subjets' Chenodeoxyholi Choli aid aid Deoxyholi aid Subjet no. HV A HV A HV A pmol/liter gmnol/liter lamollliter Hepati venous (HV) and arterial (A) blood was withdrawn simultaneously during an investigation of arbohydrate metabolism in healthy volunteers (f. 11). Bile aid onentrations were determined as desribed in Methods. that, under the present onditions, no information is ahieved with respet to the degree and nature of onjugation or sulfation of the serum bile aids. This possible limitation of the methodology used is disussed below. In the present work, fasting and postprandial levels of C, CD, and D were determined in portal venous and peripheral venous irulation of patients with holesterol gallstone disease 5-7 d after holeystetomy. Even if the patients were fully reovered from the operation, it is reasonable to assume that the bile aid pool is autely depleted by holeystetomy, and that ompensatory hanges may not have taken plae ompletely as soon as 1 wk after surgery. This probably explains why the fasting onentrations of the bile aids, partiularly CD, were somewhat lower than those previously reported (5, 7, 1). In agreement with previous work (6, 12), our study of holeystetomized subjets showed an earlier and less distint peak in peripheral serum bile aid onentrations in response to a meal ompared to what is seen in subjets with an intat gallbladder (6, 7, 12). This is presumably due to the loalization of the bile aid pool to the intestine, resulting in a more prompt response to feeding. It is reasonable to speulate that the seond peak often seen represents absorption of resereted bile aids. This study demonstrated a onsiderable inrease in portal venous bile aid onentration during a meal. The inrement in bile aid inflow to the liver is atually still higher beause the splanhni irulation is also inreased in response to feeding (13). If hepati blood flow is presumed to be 1, ml/min in the fasting state, and 1,5 ml/min during a meal, the present values would orrespond to a hepati uptake (and seretion) of -3,mol/h in fasting and maximum 728 B. Angelin, I. Bjorkhem, K. Einarsson, and S. Ewerth

6 TABLE V Estimated Hepati Frational Uptake of Individual Bile Aid Classes in Five Choleystetomized Patients" Choli aid Chenodeoxyholi aid Deoxyholi aid Patient no. Fasting Postprandial Fasting Postprandial Fasting Postprandial Mean±SEM 85±5 88±4 69±7 79±4 62±8 78±5 Calulated as.75 X P +.25 X V - Vx 1%, where P is portal venous and V peripheral venous.75 X P +.25 X V onentration. See referene 5 for details. 1,4,umol/h during a meal. The former figure is slightly lower than the alulated noturnal bile aid seretion in healthy man, 35-9 gmol/h (14-17). The latter figure is in reasonable agreement with the reported values for stimulated bile aid seretion in holeystetomized man, l1,1,umol/h (18). In spite of inreases in the portal venous bile aid onentration up to almost 6,umol/liter, we did not observe any signs of saturation of the hepati uptake system. Thus, the portal venous/peripheral venous ratio and the estimated frational uptake remained onstant-and higher for C than for CD or D-during maximal inflow of bile aids. It should be emphasized that, although the alulation of frational uptake assumes a onstant proportion between hepati arterial and portal venous blood flow, the possible error that ould be introdued by variation of this flow ratio is very limited. As mentioned above, unonjugated bile aids are not separated from onjugated with the methodology used in this work. Although the propor- A B.2 o 9 1 E - ( 3 ) o *. a W * i : o 1. ir ae Portl vwous onenra" **D1 ;:. * FIGURE 3 Relation between portal venous and peripheral venous serum onentrations of (A) holi aid, (B) henodeoxyholi aid, and (C) deoxyholi aid in five holeystetomized patients. Samples were taken before and during a standardized meal. Patient 1, who had a slightly elevated serum bilirubin level, is shown with open irles. The orrelation oeffiients (r) were (values alulated exluding patient 1 within parentheses) for holi aid +.8 (+.87), for henodeoxyholi aid +.45 (+.62), and for deoxyholi aid +.75 (+.77). The orrelations were in all ases signifiant at the P <.1 level. Systemi and portal venous onentrations (miromoles/liter). *s e. * Hepati Uptake of Bile Aids in Man 729

7 U I 6 4 E S 'I) *. o go * o eo S 2 4 Portal venous onentration FIGURE 4 Relation between portal venous and peripheral venous serum onentrations of "total bile aids" (sum of holi aid, henodeoxyholi aid, and deoxyholi aid) in five holeystetomized patients. Symbols as in Fig. 3. The orrelation oeffiient (r) was +.64 (exluding patient 1, +.73), in both ases P <.1. Systemi and portal venous onentrations (miromoles/liter). tion of unonjugated bile aids in the portal vein is probably small (f. 19), the frational uptake of these bile aids is less than that of onjugated bile aids (2). Unonjugated bile aids are always returning to the liver from the intestine, and the fration of portal bile aids in unonjugated form might be greater in holeystetomized patients. It might also be greater during the fasting state, whih may explain why, at least for D, the average frational uptake values tended to be higher in the postprandial state. Thus, the frational hepati extration estimates in Table V represent the geometri means of the hepati frational extration values for the individual speies (unonjugated, glyine onjugate, taurine onjugate) of eah of the bile aid lasses. Even with this reservation, however, it is reasonable to assume that the differene in uptake between tri- and dihydroxy bile aid lasses is related to a different affinity of the hepati transport system for different bile aids, as has been demonstrated in the rat (21), and/or to differenes in protein binding (22). The additional possibility of a differene in the lymphati transport between trihydroxy and dihydroxy bile aid has been raised by Lindblad et al. (23). However, reent studies on lymphati bile aid transport in man demonstrated that <.1% of bile aid enterohepati irulation ours via this route.2 Thus, the present results show that saturation of the hepati bile aid uptake annot be reahed under 2 Ewerth, S., I. Bjorkhem, K. Einarsson, and L. Ost J. Lipid Res. In press. 6 physiologially ourring onditions in man. This onept is in agreement with data obtained from studies in the dog (24-26) and in the rat (21, 27). The estimated frational uptake of bile aids was redued in one of our patients (No. 1). It is of interest to note, that this subjet did not display inreased levels of peripheral bile aids in the fasting or in the postprandial state. This undersores the diffiulty to identify subjets with a moderately redued hepati bile aid uptake by analysis of serum bile aids even after a load of endogenous bile aids (f. 28). A major new finding of the present work was that peripheral venous onentrations of individual bile aids reflet the simultaneous portal venous onentration. There was also a fair orrelation between total bile aids at the two sampling sites. The latter finding supports and extends the reports by Lindblad et al. (23) and Pare et al. (29). Using less speifi tehniques, these authors found parallel inreases in portal venous and peripheral venous onentrations of total bile aids in response to feeding or to holeystokinin administration in patients with malignant disease or with aloholi liver disease. The present data strongly support the onept that variation in the intestinal "input" of bile aids is the major determinant of peripheral serum bile aid onentrations in healthy man (5-7, 3). Furthermore, the portal venous inflow of bile aids an be monitored indiretly with some ertainty by analysis of peripheral venous samples. This assumption has been made in several reent studies on the enterohepati irulation of bile aids, but has never been diretly validated in man previously (f. 1). In onlusion, our study onfirms the presene of an effiient enterohepati irulation of bile aids in man. The presene of a highly effiient hepati uptake proess, whih has higher affinity for trihydroxy than for dihydroxy bile aids, and whih annot be saturated under physiologial onditions, is demonstrated. Measurement of peripheral serum bile aid onentrations reflet portal venous levels, and an thus give important information on the status of the enterohepati irulation. ACKNOWLEDGMENTS We thank Ms. Gunvor Alvelius for expert tehnial assistane, Dr. Anders Juhlin-Dannfelt and Dr. Leif Tokis for providing the hepati venous blood samples, and Ms. Lena Erisson for skillful preparation of the manusript. This study was supported by grants from the Swedish Medial Researh Counil (3X-4793), the Loo and Hans Osterman Foundation, the Swedish Soiety of Medial Sienes, and Karolinska Institutet. REFERENCES 1. Hofmann, A. F The enterohepati irulation of bile aids in man. Adv. Intern. Med. 21: B. Angelin, I. Bjdrkhem, K. Einarsson, and S. Ewerth

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